CA2342960C - G protein-coupled receptor antagonists - Google Patents

Abstract

The present invention relates to a new class of G protein-coupled receptor agonist or antagonist, which specifically binds to the receptor protein structural elements, thus altering signal transmission and subsequent physiological effects. Described herein are peptide sequences derived from the G protein-coupled receptor protein, produced by chemical methods as selective inhibitors of signal transduction associated with stimulation of the receptor by its ligand. Such peptides or molecules derived from their primary, secondary or tertiary structures may be used as effective tocolytics for the prevention of premature labor or be used for the treatment of dysmenorrhea.

Description

VV1= LY. LUUU L=LUliu unllua,a uul~,ra aua, CA 02342960 2001-03-14 = ~= J J ~ J i V, + 1 - ~ -G PROTEIN-COUPLED RECEPTOR ANTAGONISTS
BACKGROUND OF THE INVENTION

(a) Field of the Invention The invention relates to development of agonist or antagonist of a G protein-coupled receptor, which bind to the G protein-coupled receptor from the extra-cellular side in a manner different from that of the natural ligand.
(b) Descrivtion of PriQr Art Prostaglandins are derived from the oxygenation of arachidonic acid by prostaglandin synthetases. Prosta-glandins mediate a wide variety of physiological actions, such as vasomotricity, sleep/wake cycle, intestinal secretion, lipolysis, glomelular filtration, mast cell degranulation, neurotransmission, platelet aggregation, leuteolysis, myometrial contraction and labor, inflammation and arthritis, patent ductus arte-riosus, cell growth and differentiation (Coleman, R.A.
et al., 1994, Pharmacol. Rev. 46:205-229; Goetzl, E.J.
et al., 1995, FASEB J. 9:1051-10585). Prostanoids medi-ate their actions through binding to distinct recep-tors, which belong to the super family of rhodopsin-like seven transmembrane helical receptors. These receptors are coupled to heterotrimeric G-proteins com-prising of a, (3 and y subunits which, upon activation, elicit alterations in cell calcium, initiate phospho-inositide hydrolysis or promotion or repression of cyclic adenosine monophosphate synthesis (Strader C. D.
et al., 1994, Ann. Rev. Biochern. 63:101-132).
Of the five pharmacologically distinct prosta-noid receptors for PGE2, PGIzi PGDz, PGF2a, and TxA2 and their many isoforms, the receptor for PGFza, also called FP receptor, shows limited tissue distribution, pre-dominantly expressed in corpora leutea, uterine myome-

- 2 -trium, trabecular meshwork of the eye, and to a lesser extent'in vascular smooth muscle. Initiation of labor is marked by tremendous rise in PGF2p, levels and increased uterine contractility. The wide spread use of PGF2q, analogues to induce labor in veterinary industry points to the primary role of PGF2a, and its receptor in parturition. This is underscored by the fact that mice lacking'the FP receptor fail to undergo labor (Sugimoto et al., 1997, Science 277:81-83). In face of escalating costs incurred as a result of premature births and associated complications to the neonate, such as intra-ventricular hemorrhage, bronchopulmonary displasia and periventricular leukomalacia leading to cerebral palsy, prolongation of gestation by arresting premature labor is an effective preventive therapy. The relative suc-cess of nonsteroidal anti-inflammatory drugs as a short-term therapy toward prevention of premature labor is based on their inhibitory actions upon the synthesis of prostaglandins, particularly PGE2 and PGFZa. However, inhibition of PGE2 is associated with serious complica-tions to the fetus such as the closure of ductus arte-riosus, renal failure and pulmonary hypertension.
At another level, PGF2a has been attributed a major role in dysmenorrhea, a condition which afflicts 5%-7% of premenopausal women. A pre-menstrual increase in PGF2a levels resulting in myometrial spasms underlies the pathogenesis of this disorder. Lack of effective antagonists of FP receptor for extended therapy ham-pered the advances in preventing premature labor and associated sequelae, and the design of such antagonists is the subject of this application.
Human FP receptor is a 45 kDa integral membrane glycoprotein, consisting of 359 amino acids and shares only 47% sequence identity with EP1 receptor, and to a lesser extent with other prostanoid receptors

- 3 -(Abramovitz et al., 1994, J. Biol. Chem. 269:2632-26.36). Binding of PGFZa to FP receptor is followed by the activation of Gaqpy complex, increased GTP binding by the Ga,q subunit, stimulation of phospholipase C(3 activ-ity, release of inositol phosphates, increased intra-cellular calcium and subsequent signal transduction phenomena ultimately leading to smooth muscle contrac-tion (Coleman, R.A. et al., 1994, Pharmacol. Rev.
46:205-229). The FP receptor is the only efficacious target for development of therapeutic drugs since a few Ga,-proteins catalyze the actions of hundreds of G-pro-tein coupled receptors, thus targets downstream from the receptor are essentially of little use.
Antagonists of FP receptors directed to the ligand binding site could be of limited use since ligand based inhibitors show cross reactivity with other prostanoid receptors. Their efficacy will be com-promised in face of tremendous increase in PGFZa concen-trations in myometrium at the onset of labor and in menstruation. The high basal activity of the receptors in the absence of ligand limits the use of ligand-based inhibitors.
It would be highly desirable to be provided with agonist or antagonist of FP receptors, which do not crossreact with other prostanoid receptors, and are effective even in the presence of excess ligand.

SUMMARY OF THE INVENTION
One aim of the present invention is to provide agonist or antagonist of FP receptors, which do not crossreact with other prostanoid receptors.
Another aim of the present invention is to pro-vide activators or inhibitors of FP receptors by a novel strategy to target the extracellular domains of the receptor protein.

- 4 -In accordance with the present invention, there is. provided a G protein-coupled receptor agonist or antagonist which specifically binds to the juxtamem-brane extracellular structural elements of the G pro-tein-coupled receptor in a manner different from that of the natural ligand, and wherein said agonist or antagonist alter the transduction of an intracellular signal. The G protein-coupled receptor agonist or antagonist may be derived from the amino acid sequence of the receptor.
In accordance with a preferred embodiment of the present invention, the agonist or antagonist does not crossreact with other prostanoid receptors.
The antagonist is effective in the presence of excess ligand.
The agonist or antagonist may preferably com-prise an amino acid sequence derived from the first and/or second extracellular loops of prostanoid recep-tors.
In accordance with another embodiment of the present invention, the antagonists of the present invention comprise amino acid sequences derived from the first and second extracellular loops of prostanoid receptors.
In accordance with a preferred embodiment of the present invention, the G protein-coupled receptor is the prostaglandin F2,,, receptor (FP receptor).
In accordance with a preferred embodiment of the present invention, the antagonist of the present inven-tion comprises amino acid sequences derived from the prostaglandin F2a receptor.
Preferably, the antagonist include, without limitation, amino acid sequence of the FP receptor selected from the group consisting of ILGHRDYK (PCP-8;
SEQ ID NO:1); WEDRFYLL (PCP-10; SEQ ID NO:2); YQDRFYLL

- 5 -(PCP-14; SEQ ID NO:3); ILAHRDYK (PCP-13.7; SEQ ID
NO:4); 'ILGFRDYK (PCP-13.11; SEQ ID NO:5); ILGHKDYK
(PCP-13.13; SEQ ID NO:6) ; ILGHRNYK (PCP-13.14; SEQ ID
NO:7); ILGHQDYK (PCP-13.18; SEQ ID NO:8); ILGHRDY-amide (PCP-13.20; SEQ ID NO:9); ILGHRDYK-amide (PCP-13.21;
SEQ ID NO:1); ILGWRDYK (PCP-13.22; SEQ ID NO:10);
ILGXRDYK (PCP-13.24; SEQ ID NO:11); SNVLCSIF (PCP-15;
SEQ ID NO:12) protein fusions and peptidomimetics thereof; wherein said amino acid sequence contains L-and/or D-amino acid.
In accordance with the present invention, there is provided a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l to 12 and wherein said amino acid sequence contains L- and/or D-amino acid, an amino acid sequence with at least about 90% homology to SEQ ID NO:1 to 12, and peptidomi-metic thereof.
In accordance with the present invention, there is provided a pharmaceutical composition containing at least a G protein-coupled receptor agonist and antago-nist of the present invention, mixture thereof, or functional derivatives thereof in association with a pharmaceutically acceptable carrier.
In accordance with another embodiment of the present invention, there is provided a method for pre-venting premature delivery of fetus, which comprises the step of administering to a female in need of such a treatment a therapeutically effective amount of a G
protein-coupled receptor antagonist or functional derivatives thereof, wherein the antagonist or func-tional derivatives thereof specifically binds to the extracellular face of the receptor, thereby hampering uterine contractions.
In accordance with another embodiment of the present invention, there is provided a method for pre-

- 6 -venting and/or treating dysmenorrhea comprising the step of administering to a female in need of such a treatment a therapeutically effective amount of a G
protein-coupled receptor antagonist or functional derivatives thereof, wherein the antagonist or func-tional derivatives thereof specifically binds to the extracellular face of the receptor to hamper transduc-tion of a signal thereby reducing the pain associated with contractions.
In accordance with another embodiment of the present invention, there is provided a method of iden-tifying a compound as a G protein-coupled receptor ago-nist or antagonist capable of binding to the extracel-lular elements of the said receptor in a manner differ-ent from that of the natural ligand, comprising the steps of:
a) culturing cells which express said receptor or identifying animal tissues ex vivo or in vivo where physiological consequences are dependent on said receptor;
b) contacting said cells or tissues with said com-pound to be tested for agonist or antagonist activity at said receptor; and c) measuring a response to alter the transduction of a signal resulting in physiological conse-quences selected from the group consisting of increments in cell calcium, phosphoinositide hydrolysis, increased/decreased cellular cyclic adenosine monophosphate, cell growth and/or dif-ferentiation, altered gene expression, and smooth muscle contraction or dilation, wherein said response is indicative of agonist or antagonist activity.
In accordance with another embodiment of the present invention, there is provided a method of iden-

- 7 -tifying a compound as a prostaglandin F2 alpha receptor agonist' or antagonist capable of binding to the extra-cellular elements of the said receptor in a manner dif-ferent from that of the natural ligand, comprising the steps of:
a) culturing cells which express said receptor or identifying animal tissues ex vivo or in vivo where physiological consequences are dependent on said receptor;
b) contacting said cells or tissues with said com-pound to be tested for agonist or antagonist activity at said receptor; and c) measuring a response to alter the transduction of a signal resulting in physiological conse-quences selected from the group consisting of increments in cell calcium, phosphoinositide hydrolysis, cell growth and/or differentiation, altered gene expression, and smooth muscle con-traction or dilation, wherein said response is indicative of agonist or antagonist activity.
For the purpose of the present invention the following terms are defined below.
The expression "a G protein-coupled receptor agonist or antagonist" is intended to mean any natural or synthetic compound, peptide protein, antibody, pep-tidomimetic or small chemical molecules, without limi-tation, insofar as it can specifically bind to the extracellular structural elements of the G protein-cou-pled receptor to alter transduction of a signal. More preferably, the agonist or antagonist does not crossre-act with other prostanoid receptors.
The expression "functional derivatives" of G
protein-coupled receptor agonist or antagonist is intended to mean mimetic compounds and/or structurally unrelated compounds with respect to G protein-coupled

8 PCT/CA99/00844 receptor antagonist, which can also specifically bind to the- extracellular structural elements of the G pro-tein-coupled receptor to alter transduction of a sig-nal.
The expression "peptidomimetic thereof" is intended to mean any chemical entities, mimetic com-pounds and/or structurally unrelated compounds with respect to G protein-coupled receptor agonist or antagonist, which can also specifically bind to the extracellular structural elements of the G protein-cou-pled receptor to alter transduction of a signal.

In an aspect, the present invention provides a prostaglandin F2 receptor antagonist which comprises an amino acid sequence of a prostaglandin F2 receptor, said amino acid sequence consisting of one or more sequences selected from the group consisting of ilghrdyk (PCP-8 SEQ ID NO:1), ILGHRDYK (PCP-13; SEQ ID
NO:13); WEDRFYLL (PCP-10; SEQ ID NO:2); YQDRFYLL (PCP-14; SEQ ID NO:3); ILAHRDYK (PCP-13.7; SEQ ID NO:4);
ILGFRDYK (PCP-13,11; SEQ ID NO:5); ILGHKDYK (PCP-13.13; SEQ ID NO:6); ILGHRNYK (PCP-13.14; SEQ ID
NO:7); ILGHQDYK (PCP-13.18; SEQ ID NO:8); ILGHRDY-amide (PCP-13.20; SEQ ID NO:9); ILGHRDYK-amide (PCP-13.21; SEQ ID NO:15); ILGWRDYK (PCP-13.22; SEQ ID
NO:10); ILGXRDYK (PCP-13.24; SEQ ID NO:11) wherein X
is cyclohexyl alanine; SNVLCSIF (PCP-15; SEQ ID NO:12) and ILaHRDYK (PCP-13.8; SEQ ID NO:14), and wherein small letters indicate L-amino acids and capital letters indicate D-amino acids.

Ba The present invention further provides a peptide consisting of:
(a) an amino acid sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8,

9, 10, 11, 12, 13, 14 and 15, wherein said amino acid sequence contains L-amino acids, D-amino acids, or both; or (b) an amino acid sequence with at least 900 sequence identity to at least one of SEQ ID NO:1 to 15 and having the biological activity of a sequence of at least one of SEQ ID NO:1 to 15.
The present invention further provides a peptide consisting of a variant sequence of SEQ ID NO:1 in which one or two amino acid residues are substituted or deleted, wherein said variant sequence contains one or both L- and D-amino acids and wherein said peptide is a prostaglandin F2 receptor antagonist.
The present invention further provides a peptide consisting of a variant sequence of SEQ ID NO:1 in which one or two amino acid residues are substituted or deleted, wherein said variant sequence contains one or both L- and D-amino acids and a conversion of a C-terminal CO2H group to a CONHZ group, and wherein said peptide is a prostaglandin F2 receptor antagonist.
The present invention further provides a pharmaceutical composition comprising at least one antagonist mentioned above, in association with a pharmaceutically acceptable carrier.
The present invention further provides a pharmaceutical composition comprising at least one peptide mentioned above in association with a pharmaceutically acceptable carrier.

8b The present invention further provides a method for determining activity of the above-mentioned antagonist as a prostaglandin F2 alpha receptor antagonist capable of binding to extracellular elements of the said receptor, comprising the steps of:
a) culturing cells which express said receptor or identifying animal tissues ex vivo or in vivo where physiological consequences are dependent on said receptor;
b) contacting said cells or tissues with said compound at a concentration of 10-10 M to 10-3 M to be tested for antagonist activity at said receptor; and c) measuring a response to alter the transduction of a signal resulting in physiological consequences selected from the group consisting of increments in cell calcium, phosphoinositide hydrolysis, cell growth, differentiation, altered gene expression, and smooth muscle contraction or dilation.
The invention further provides a use of a therapeutically effective amount of an antagonist mentioned above for the preparation of a medicament for preventing premature delivery of a fetus.
The invention further provides a use of a therapeutically effective amount of an antagonist mentioned above for the preparation of a medicament for preventing or treating dysmenorrhea.
The invention further provides a use of a therapeutically effective amount of an antagonist mentioned above for the preparation of a medicament for decreasing the likelihood of premature delivery.

8c The invention further provides a use of a therapeutically effective amount of an antagonist mentioned above for the preparation of a medicament for reducing uterine contractions.
The invention further provides a use of a therapeutically effective amount of a peptide mentioned above for the preparation of a medicament for preventing premature delivery of a fetus.
The invention further provides a use of a therapeutically effective amount of a peptide mentioned above for the preparation of a medicament for preventing, reducing the occurrence of, or treating dysmenorrhea.
The invention further provides a use of a therapeutically effective amount of a peptide mentioned above for the preparation of a medicament for decreasing the likelihood of premature delivery.
The invention further provides a use of a therapeutically effective amount of a peptide mentioned above for the preparation of a medicament for reducing uterine contractions.
The invention further provides a use of an antagonist mentioned above for preventing premature delivery of a fetus.
The invention further provides a use of an antagonist mentioned above for preventing or treating dysmenorrhea.
The invention further provides a use of an antagonist mentioned above for decreasing the likelihood of premature delivery.
The invention further provides a use of an antagonist mentioned above for reducing uterine contractions.

8d The invention further provides a use of a peptide mentioned above for preventing premature delivery of a fetus.

The invention further provides a use of a peptide mentioned above for preventing, reducing the occurrence of, or treating dysmenorrhea.
The invention further provides a use of a peptide mentioned above for decreasing the likelihood of premature delivery.

The invention further provides a use of a peptide mentioned above for reducing uterine contractions.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 illustrates the inhibitory effects of PCP-8 and PCP-10 on FP receptor function upon stimula-tion with PGF2CI in accordance with the embodiment of the present invention;
Fig. 2A illustrates the effects of PCP-8 and PCP-10 on the diameter of the microvessels of pig retina upon stimulation with either PGF2Q or thromboxane A2 mimetic, U46619;
Fig. 2B illustrates the dose response of PGF2Q
on the diameter of pig microvessels treated previously with PCP-8 or PCP-10;
Fig. 2C illustrates the effects of thromboxane A2 mimetic, U46619, on the diameter of pig microvessels treated previously with PCP-8 and PCP-10;
Fig. 3A illustrates the effects of PCP-10 upon spontaneous contractions of uterine smooth muscle;
Fig. 3B illustrates the dose response of prosta-glandin Fza in the presence/absence of PCP-8 and_PCP-10 upon uterine smooth muscle contraction; and Fig. 4 illustrates the reversal of basal tone of bovine myometrium even in the presence of FP receptor ligand, PGFZQ.

DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, there is provided a new class of G protein-coupled receptor antagonists, which bind to the extracellular molecular surface, thus hamper signal transduction.
Also provided is a novel strategy to target the extracellular loops of the receptor which contribute to the structural or functional integrity of the receptor.
Antagonists thus bind to cognate elements in the extra-cellular surface of the receptor and prevent the recep-tor function by interfering with its signal initiation or transduction.
There is provided proof of selectivity of the antagonists to FP receptor by showing an absence of their effects on a related prostanoid receptor for thromboxane A2, known as TP receptor which is also involved in smooth muscle contraction.
Preparation of inhibitors Chemical synthesis of PCP-8 and PCP-10:
All peptides which are 8 amino acids in length were synthesized using F-moc chemistry and solid phase Merrifield method two peptides, PCP-8 and PCP-10. These peptides were purified by HPLC and their purity tested by mass spectroscopy.
In accordance with the present invention, a novel strategy of using peptides derived from the extracellular domains of prostaglandin F2a receptor, FP, to inhibit the signal transduction and the functional consequences of FP receptor. This method could be gen-eralized to all G protein-coupled receptors. Peptides derived from the first and second extracellular loops of FP receptor were found to be effective inhibitors of FP receptor.

- 10 -The present invention could be readily under-.
stood by referring to the following examples, which are given to illustrate the invention rather than to limit its scope.
EXAMPLE I
Effects of peptides, PCP-8 and PCP-10, on ligand-induced phosphoinositide hydrolysis in mammalian cells overexpressing the FP receptor Both PCP-8 and -10 were tested in HEK293 cells expressing the human FP receptor. For this purpose, HEK
293 cells stably expressing human FP receptor were plated in 12-well plates in DMEM medium containing 10%
fetal bovine serum, penicillin (10 U/ml) and streptomy-cin (10 g/ml) and cultured in a humidified atmosphere containing 5% C02 at 37 C. After the wells were 80% con-fluent, the cells were labeled with 2 Ci/ml of [3H]-myo inositol overnight. Next day, the cells were washed once with PBS, and incubated in 0.5 ml of Kreb's buffer containing 10 mM LiCl and indicated concentrations of PCP peptides for 30 min. PGFZa at 1 M was added to the cells and the incubation was carried out for an addi-tional 30 min. The cells were solubilized with 0.1 N
NaOH for 10 min and neutralized with 0.1 N formic acid.
The lysates were collected and 1 ml each of methanol and chloroform were sequentially added and vortexed briefly. After centrifugation to separate the phases, inositol phosphates were separated by ion exchange chromatography as described below (Berridge, M.J. et al., 1983, Biochem. J. 212:473-482).
Briefly, the medium was discarded and the IP3 synthesis was stopped by adding 0.6 ml ice-cold metha-nol. The cells were scraped and collected into polypro-pylene tubes. Distilled water (0.5 ml) and chloroform (0.6 ml) were added and vigorously vortexed for 2 min.
The phases were separated by centrifugation at 6000 x g

- 11 -for 10 min. The aqueous phase was applied to AG-1X-8T"' (Format'e form) anion exchange columns (1 ml bed volume) and free inositol was eluted with 10 ml of water, fol-lowed by 60 mM ammonium formate in 0.1 M formic acid.
Then, the inositol phosphates were eluted with 5 ml of 1.2 M ammonium formate in 0.1 M formic acid. After add-ing 3 volumes of scintillation cocktail (Optiphase-HiSafe IIITM), the eluates were counted by scintillation spectrophotometry.
The results of these experiments are shown in Fig. 1. Data are expressed as fold stimulation of inositol phosphate synthesis by 1 M PGF2a compared to the unstimulated controls. Both PCP-8 and -10 at 100 M
potently inhibited inositol phosphate synthesis initi-ated by the action of PGFZa on FP receptor. The half maximal inhibitory concentrations for both PCP-8 and -10 were slightly less than 100 M.

EXAMPLE II
Testing PCP peptides in porcine eyecup model of ex vivo vasomotricity assay In order to see if the peptides could inhibit FP
function using an ex vivo model, we chose porcine eye-cup model, an ex vivo assay of vascular constriction in porcine retinas which we previously described and vali-dated (Li et al., 1996 J. Pharmacol. Expt. Therapeut.
278: 370-377; Li et al., 1997 Am. J. Physiol. 273:
R1283-90; Abran et al., 1997 Am. J. Physiol. 272: R995-1001) . Since FP receptor densities in newborn vascula-ture are minimal due to down regulation by high levels of circulating prostaglandins in the perinatal period, the newborn pigs were treated with a prostaglandin syn-thetase blocker, ibuprofen (30 mg/Kg of bodyweight/ 8 h for 24 h) to increase the density of the receptors as well as their vasomotor effects. By inhibiting circu-lating prostaglandins, we were able to show potent

- 12 -inhibition of FP receptor-mediated second messenger synthesis as well as FP-mediated vascular constriction in this eyecup model.
To prepare eyecups, a circular incision was made 3-4 mm posterior to ora serrata to remove the interior segment and vitreous body with minimal handling of the retina. The remaining eyecup was fixed with pins to a wax base in a 20 ml tissue bath containing 20 ml of Kreb's buffer (pH 7.35-7.45), protease inhibitors, leu-petin and aprotinin (10 g/ml each), and equilibrated with 21% oxygen and 5% carbon dioxide at 37 C. The preparations were allowed to stabilize for 30 min. Pep-tides at 100 M were added and incubation was continued for 30 min before the addition of PGF2a.
Cumulative concentration-responses of PGF2a and TxA2 mimetic, U46619, (10-10 to 10-5 M) curves were con-structed separately. To assess the reversibility of the antagonists, the eyecups were thoroughly washed (which would wash away the peptide) with incubation medium and concentration response curves for PGF21 were determined.
The outer vessel diameter was recorded with a video camera mounted on a dissecting microscope (Zeiss M
4OOTM) and the responses were quantified by a digital image analyzer (Sigma Scan Software, Jandel Scientific, Corte Madera, CA). Vascular diameter was recorded before and 10 min following the topical application of the agent. Each measurement was repeated three times and showed <1% variability.
The results are shown in Fig. 2. The peptide PCP-10 had no effect on the basal tone (diameter of the microvessel) of the vessel (Fig. 2A; left panels).
Addition of 1 M of PGF2a potently constricted the ves-sel in the absence of the peptide (middle-top panel), whereas presence of PCP-10 at 100 M markedly inhibited PGF2a-mediated vasoconstriction (middle-bottom panel).

- 13 -The peptide had no effect on the vasoconstriction effecte'd by 1 M TxA2 mimetic, U46619, (right panels) acting on another prostanoid receptor coupled to con-striction, namely TP receptor. Similar results were obtained for PCP-8 as well. A dose response of PGFza on the vascular diameter in the presence/absence of PCP-8 and PCP-10 peptides are presented in Fig. 2B. Both pep-tides abrogated the vasomotor responses even at concen-trations exceeding 1 M of PGFza, suggesting, as expected, that the peptides may be acting in a non-com-petitive fashion. However, the peptides had no effect on vasoconstriction produced by thromboxane A2 (Fig.
2C).
Similarly, a peptide derived from the first extracellular loop of FP receptor, PCP-15, inhibited PGF2a,-induced constriction (10-'M) (88.1% over untreated control; Table 1).

EXAMPLE III
Testing peptide variants of PCP-8 in porcine eyecup model of ex vivo vasomotricity assay In order to understand the structural requirements of PCP-8 in its inhibitory action on PGF2ainduced vaso-constriction, different amino acids in PCP-8 sequence were replaced with other D- or L- amino acids and the resulting peptides were chemically synthesized and tested in porcine eyecup model of ex vivo vasomotricity assay. These peptide variants are listed in Table 1.

WO 00/1?348 PCT/CA99/00844

- 14 -Table 1 Amino acid sequences of peptide variants of PCP-8 and their inhibitory potency in porcine eyecup model of ex vivo vasomotricity assay Peptide %Vasomotor /. inhibition Peptide sequence SEQ ID NO:
PCP- response (of of maximal max. response 2 constriction ' 8 50.0 50.0 il hrd k I
20.0 80.0 wed Il 2 14 36.0 64.0 YQDRFYLL 3 13 20.0 80.0 ILGHRDYK 13 13.7 23.8 76.2 ILAHRDYK 4 13.8 46.8 53.2 lLaHRDYK 14 13.11 13.0 87.0 ILGFRDYK 5 13.13 36.9 63.1 ILGHKDYK 6 13.14 40.3 59.7 ILGHRNYK 7 13.18 30.0 70.0 ILGHQDYK 8 13.20 49.6 50.4 ILGHRDY-amide 9 13.21 46.2 53.8 ILGHRDYK-amide 15 13.22 16.6 83.4 ILGWRDYK 10 13.24 6.2 93.8 ILGXRDYK 11 11.9 88.1 SNVLCSIF 12 5 'Percent vasomotor response in the presence of 100 M
peptide is calculated as percent change in average vascu-lar diameter produced by 10"' M PGFZa to the eyecup in the presence of the peptide compared to maximal constriction observed in the absence of the peptide.
10 2 Percent inhibition produced by each peptide is calcu-lated as (100-per cent vasomotor response).

Small letters indicate L-amino acids and capital letters indicate D- amino acids. I = isoleucine; L=

15 leucine; G =glycine; H=histidine; R=Arginine;
D=Aspartic acid; Y=Tyrosine; K=Lysine; A=Alanine; W=
Tryptophan; E=Glutamic acid; F= Phenyl alanine;
Q=Glutamine; N=Aspargine; P=Proline; S=Serine;
X=Cyclohexyl alanine. Peptides were dissolved in DMSO
freshly just before the experiment as 10 mM stocks and added to the eye cups 30 min before the addition of 10'' M PGF2a.

A total of 25 variants of PCP-8 were synthesized and the efficacious or potent peptides are listed in Table 1. These peptides incorporate L- to D-amino acid changes, deletions, subtle variations in aromaticity, hydrogen bond donor status as opposed to ionic interac-tions and hydrophobicity. These peptides were tested at 100 M concentration in porcine retinal vasomotricity assay and the results are summarized in Table 1.
The results are summarized as follows:
1. Converting all L-amino acids of PCP-8 to D-amino acids (PCP-13) increased the inhibitory potency dra-matically. Removal of N-terminal hydrophobic dipep-tide sequence from either PCP-8 (PCP-11) or PCP-10 (PCP-12) resulted in significant reduction in the inhibitory action of the peptides.
2. Glycine to alanine (13.7) does not change the activ-ity of PCP-13, whereas change to proline (13.16), L-alanine (13.8), or deletion of the residue (13.17) entirely resulted in loss of activity. Glycine is an important linker residue separating the HRD motif from the IL hydrophobic sequence.
3. HRD-motif is important for the activity of PCP-13.
Alanine substitutions (13.1-13.3) or to change to L-configuration (13.4-13.6) resulted loss of inhibitory activity of PCP-13. Aromaticity of His is more impor-tant than the positive charge, since H to F (13.11) or W (13.22) or X (13.24), but not to Y (13.23), did not result in significant reduction of peptide inhibitory potency. Side chain length appears to be more critical in case of D residue than R; changing D
to E (13.12) resulted in loss of half of the inhibi-tory activity whereas R to K (13.13) or to Q (13.18) affected the activity of PCP-13 moderately. D to N
(13.14) resulted in moderate loss of activity,

- 16 -whereas a serine substitution (13.19) lead to drastic loss'of activity of PCP-13.
4. Deletion of terminal lysine (13.15) or substitution with W (13.9) resulted in complete loss of activity;
however, conversion of terminal carboxylate into an amide (13.20 & 13.21) resulted in moderate gain of activity of the peptide inhibitor. Substitution of aromatic residue, Y, with E (13.10) completely abol-ished the inhibitory potency of PCP-13.

Thus the structure of PCP-13 in D-configuration appears to consists of a N-terminal hydrophobic anchor spaced from the central HRD motif by a glycine residue possibly resulting in a turn conformation of the active peptide; Aromatic and hydrophobic interactions at the carboxy terminus may also add to the potency of PCP-13.
EXAMPLE IV
Testing PCP peptides in porcine uterine strip of ex vivo basal contraction assay In ex vivo experiments using porcine uterine strips, the peptides were able to prevent both basal and PGF2a,-induced contraction.
Uterine tissues from non-pregnant adult pigs were obtained from a local slaughter house and trans-ported to the laboratory on ice. Uterine myometrial strips of approximately 1 cm in length were set up in organ baths containing Kreb's buffer equilibrated with 21% oxygen at 37 C as we have described (Potvin, W. et al., 1990, Br. J. Pharmacol. 100:341-347; Varma, D.R.
and Chemtob, S., 1993, J. Pharmacol. Expt. Ther.
265:1096-1104). Contractions were recorded by force transducers on Grass-polygraph. Strips were incubated with or without 100 M peptides for 30 min before add-ing PGF2a in step-wise increments (10-9 to 10-6 M) . Data were expressed as percentage increase over the basal level of average tension (g).

- 17 -A graph of spontaneous uterine contractions (known -to be dependent upon prostanoids, mainly PGF2a) in the absence and the presence of 100 M PCP-8 are shown in Fig. 3A. Addition of peptide to the strips reduced the force of basal contraction. A dose response of PGF21 on uterine contractility in the presence or absence of PCP-8 and PCP-10 peptides is shown in Fig. 3B. More than 60% (PCP-8) and 80% (PCP-10) reduc-tion in uterine contraction was observed in all concen-trations of PGF2a, tested. Thus, both these peptides reduced spontaneous as well as PGF2a,-induced contrac-tions in the uterine strips.

EXAMPLE V
Testing PCP peptides in bovine uterine strip of ex vivo basal contraction assay Uterine tissues from non-pregnant adult bovine animals were obtained from a local slaughter house and transported to the laboratory on ice. Uterine myome-trial strips of approximately 1 cm in length were set up in organ baths containing Kreb"s buffer equilibrated with 21% oxygen at 37 C as described above. Contrac-tions were recorded on Grass-polygraph by force trans-ducers. Strips were incubated with or without 100 M
peptides before adding PGF2a in step-wise increments (10-$ to 10-6 M) . Data were expressed as change in basal level of average tension (g) . The results are shown in Fig. 4. Application of PCP-10 peptide at 100 M
reversed the basal tone (contractile state) of the uterine muscle. Addition of PGFZa up to lO M did not affect the relaxation produced by PCP-10 suggesting that the effects of PCP peptides are independent of the ligand, which was also shown in the previous results.
While the invention has been described in con-nection with specific embodiment thereof, it will be understood that it is capable of further modifications

- 18 -and this application is intended to cover any varia-tions,' uses or adaptations of the invention following in general, the principles of the invention and includ-ing such departures from the present disclosure as come within the known customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

18a SEQUENCE LISTING
<110> HOPITAL SAINTE-JUSTINE

<120> G PROTEIN-COUPLED RECEPTOR AGONISTS OR ANTAGONISTS
<130> 780/11718.205 <140> CA 2,342,960 <141> 1999-09-15 <150> US 09/154,627 <151> 1998-09-17 <160> 15 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 8 <212> PRT
<213> Artificial Sequence <220>
<223> peptide antagonist derived from the sequence of the prostaglandin F2-alpha receptor <400> 1 Ile Leu Gly His Arg Asp Tyr Lys <210> 2 <211> 8 <212> PRT
<213> Artificial Sequence <220>
<223> peptide antagonist derived from the sequence of the prostaglandin F2-alpha receptor <400> 2 Trp Glu Asp Arg Phe Tyr Leu Leu <210> 3 <211> 8 <212> PRT
<213> Artificial Sequence <220>
<223> peptide antagonist derived from the sequence of the prostaglandin F2-alpha receptor 18b <221> SITE
<222> (1)...(8) <223> peptide containing all D-amino acids <400> 3 Tyr Gln Asp Arg Phe Tyr Leu Leu <210> 4 <211> 8 <212> PRT
<213> Artificial Sequence <220>
<223> peptide antagonist derived from the sequence of the prostaglandin F2-alpha receptor <221> SITE
<222> (1)...(8) <223> peptide containing all D-amino acids <400> 4 Ile Leu Ala His Arg Asp Tyr Lys <210> 5 <211> 8 <212> PRT
<213> Artificial Sequence <220>
<223> peptide antagonist derived from the sequence of the prostaglandin F2-alpha receptor <221> SITE
<222> (1)...(8) <223> peptide containing all D-amino acids <400> 5 Ile Leu Gly Phe Arg Asp Tyr Lys <210> 6 <211> 8 <212> PRT
<213> Artificial Sequence <220>
<223> peptide antagonist derived from the sequence of the prostaglandin F2-alpha receptor <221> SITE
<222> (1)...(8) <223> peptide containing all D-amino acids <400> 6 Ile Leu Gly His Lys Asp Tyr Lys <210> 7 <211> 8 <212> PRT
<213> Artificial Sequence <220>
<223> peptide antagonist derived from the sequence of the prostaglandin F2-alpha receptor <221> SITE
<222> (1)...(8) <223> peptide containing all D-amino acids <400> 7 Ile Leu Gly His Arg Asn Tyr Lys <210> 8 <211> 8 <212> PRT
<213> Artificial Sequence <220>
<223> peptide antagonist derived from the sequence of the prostaglandin F2-alpha receptor <221> SITE
<222> (1)...(8) <223> peptide containing all D-amino acids <400> 8 Ile Leu Gly His Gln Asp Tyr Lys <210> 9 <211> 7 <212> PRT
<213> Artificial Sequence <220>
<223> peptide antagonist derived from the sequence of the prostaglandin F2-alpha receptor 18d <221> SITE
<222> (1)...(7) <223> peptide containing all D-amino acids <221> AMIDATION
<222> (7)...(7) <223> The OH group of the Tyrosine at position 7 has been replaced with an NH2 group <400> 9 Ile Leu Gly His Arg Asp Tyr <210> 10 <211> 8 <212> PRT
<213> Artificial Sequence <220>
<223> peptide antagonist derived from the sequence of the prostaglandin F2-alpha receptor <221> SITE
<222> (1)...(8) <223> peptide containing all D-amino acids <400> 10 Ile Leu Gly Trp Arg Asp Tyr Lys <210> 11 <211> 8 <212> PRT
<213> Artificial Sequence <220>
<223> peptide antagonist derived from the sequence of the prostaglandin F2-alpha receptor <221> SITE
<222> (1)...(8) <223> peptide containing all D-amino acids <221> SITE
<222> (4)...(4) <223> Xaa at position 4 = cyclohexyl alanine <400> 11 Ile Leu Gly Xaa Arg Asp Tyr Lys 18e <210> 12 <211> 8 <212> PRT
<213> Artificial Sequence <220>
<223> peptide antagonist derived from the sequence of the prostaglandin F2-alpha receptor <221> SITE
<222> (1)...(8) <223> peptide containing all D-amino acids <400> 12 Ser Asn Val Leu Cys Ser Ile Phe <210> 13 <211> 8 <212> PRT
<213> Artificial Sequence <220>
<223> peptide antagonist derived from the sequence of the prostaglandin F2-alpha receptor <221> SITE
<222> (1)...(8) <223> peptide containing all D-amino acids <400> 13 Ile Leu Gly His Arg Asp Tyr Lys <210> 14 <211> 8 <212> PRT
<213> Artificial Sequence <220>
<223> peptide antagonist derived from the sequence of the prostaglandin F2-alpha receptor <221> SITE
<222> (1)...(2) <223> the amino acids at positions 1 and 2 are D-amino acids <221> SITE
<222> (4)...(8) <223> the amino acids at positions 4 to 8 are D-amino 18f acids <400> 14 Ile Leu Ala His Arg Asp Tyr Lys <210> 15 <211> 8 <212> PRT
<213> Artificial Sequence <220>
<223> peptide antagonist derived from the sequence of the prostaglandin F2-alpha receptor <221> AMIDATION
<222> (8)...(8) <223> The OH group of the Lysine at position 8 has been replaced with an NH2 group <221> SITE
<222> (1)...(8) <223> peptide containing all D-amino acids <400> 15 Ile Leu Gly His Arg Asp Tyr Lys

Claims (9)

WHAT IS CLAIMED IS:

1. A prostaglandin F2 receptor antagonist which comprises an amino acid sequence of a prostaglandin F2 receptor, said amino acid sequence consisting of one or more sequences selected from the group consisting of ilghrdyk (PCP-8 SEQ
ID NO:1), ILGHRDYK (PCP-13; SEQ ID NO:13); WEDRFYLL (PCP-10; SEQ ID NO:2); YQDRFYLL (PCP-14; SEQ ID NO:3); ILAHRDYK
(PCP-13.7; SEQ ID NO:4); ILGFRDYK (PCP-13,11; SEQ ID NO:5);
ILGHKDYK (PCP-13.13; SEQ ID NO:6); ILGHRNYK (PCP-13.14; SEQ
ID NO:7); ILGHQDYK (PCP-13.18; SEQ ID NO:8); ILGHRDY-amide (PCP-13.20; SEQ ID NO:9); ILGHRDYK-amide (PCP-13.21; SEQ ID
NO:15); ILGWRDYK (PCP-13.22; SEQ ID NO:10); ILGXRDYK (PCP-13.24; SEQ ID NO:11) wherein X is cyclohexyl alanine;
SNVLCSIF (PCP-15; SEQ ID NO:12) and ILaHRDYK (PCP-13.8; SEQ
ID NO:14), and wherein small letters indicate L-amino acids and capital letters indicate D-amino acids.

2. A peptide consisting of:
(a) an amino acid sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15, wherein said amino acid sequence contains L-amino acids, D-amino acids, or both; or (b) an amino acid sequence with at least 90% sequence identity to at least one of SEQ ID NO:1 to 15 and having the biological activity of a sequence of at least one of SEQ ID NO:1 to 15.

3. A pharmaceutical composition comprising at least one antagonist of claim 1, in association with a pharmaceutically acceptable carrier.

4. A pharmaceutical composition comprising a peptide of claim 2 in association with a pharmaceutically acceptable carrier.

5. A method for determining activity of an antagonist of claim 1 as a prostaglandin F2 alpha receptor antagonist capable of binding to extracellular elements of said receptor, comprising the steps of:
a) culturing cells which express said receptor or identifying animal tissues ex vivo or in vivo where physiological consequences are dependent on said receptor;
b) contacting said cells or tissues with said compound at a concentration of 10 -10 M to 10 -3 M to be tested for antagonist activity at said receptor; and c) measuring a response to alter the transduction of a signal resulting in physiological consequences selected from the group consisting of increments in cell calcium, phosphoinositide hydrolysis, cell growth, differentiation, altered gene expression, and smooth muscle contraction or dilation.

6. Use of a therapeutically effective amount of an antagonist of claim 1 for the preparation of a medicament for reducing uterine contractions.

7. Use of a therapeutically effective amount of a peptide of claim 2 for the preparation of a medicament for reducing uterine contractions.

8. Use of an antagonist of claim 1 for reducing uterine contractions.

9. Use of a peptide of claim 2 for reducing uterine contractions.